Spizellomyces punctatus

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Spizellomyces punctatus
Scientific classification edit
Kingdom: Fungi
Division: Chytridiomycota
Class: Chytridiomycetes
Order: Spizellomycetales
Family:
Genus: Spizellomyces
Species:
S. punctatus
Binomial name
Spizellomyces punctatus
(W.J. Koch) D.J.S. Barr

Spizellomyces punctatus is a chytrid fungal species that is found in soil.[1] It is a saprotrophic fungus that colonizes decaying plant material.[2] Being an early diverging fungus, S. punctatus retains ancestral cellular features that are also found in animals and amoebae.[3] Its pathogenic relatives, Batrachochytrium dendrobatidis and B. salamandrivorans, infect amphibians and cause global biodiversity loss.[4] The pure culture of S. punctatus was first obtained by Koch (named Phlyctochytrium punctatum).[5]

Genome[]

The genome of S. punctatus strain DAOM BR117 was sequenced under the Origins of Multicellularity project.[6] Its genome size is about 24.13 Mb with a GC content of 47.6%. The genome harbors 9,424 predicted transcripts and 8,952 predicted protein-coding genes. The DDBJ/EMBL/GenBank accession number is ACOE00000000.[7]

Genetic transformation[]

Agrobacterium-Mediated Transformation[]

Genetic transformation of S. punctatus zoospores by plant pathogen Agrobacterium tumefaciens EHA105 strain is successfully established. Several selection markers have been tested. The growth of S. punctatus is not inhibited by Geneticin (G418), Puromycin, and Phleomycin D10 (Zeocin) up to 800 mg/L. 200 mg/L Hygromycin and 800 mg/L Nourseothricin (CloNAT) completely inhibit S. punctatus growth. The scientists who develop this protocol use Hygromycin as the selection marker. S. punctatus HSP70 and H2B promoters drive sufficient gene expression for Hygromycin resistance and GFP expression tested in yeast. Controlled by stronger H2B promoter, however, GFP may not be successfully folded in S. punctatus. Other fluorescent proteins, including tdTomato, mClover3, mCitrine, and mCerulean3, are functional in S. punctatus.[3]

Electroporation[]

A high-efficiency electroporation protocol for S.punctatus and two related chytrids species B. dendrobatidis and B. salamandrivorans has also been established. The optimal voltage for S. punctatus is 1000 V. The efficiency is about 95% using synchronized zoospores. Electroporation using unsynchronized zoospores can also reach more than 80% efficiency.[8]

Life cycle[]

S.punctatus globular zoospores (3–5 mm) lacks a cell wall. The zoospores can swim with a motile cilium (20–24 mm) or crawl on surfaces by actin-filled pseudopods.[3]

During encystment, the cilium is disassembled first via axoneme internalization. The initiation of this process is actin-dependent. The axoneme remains intact during internalization and the axonemal tubulin is degraded at least in part by the proteasome. The cell wall is formed after axoneme internalization. Five modes of axoneme internalization occur in S. punctatus: severing, reeling in retraction, lash-around retraction, ciliary compartment loss retraction, and vesicular retraction. First, severing is referred to as cilium detachment. Second, reeling in retraction is concurrent with or without cortical rotation and termed body-twist retraction and straight-in retraction, respectively. Third, during lash-around retraction, the cilium wraps around outside the zoospore with merging of ciliary membrane and plasma membrane. On 120 kPa fibronectin-coated hydrogels, this lash-around retraction occurs within a second. Fourth, for ciliary compartment loss retraction, ciliary membrane expansion is followed by merging of the ciliary compartment with the plasma membrane. Fifth, vesicular retraction is the creation of an axoneme loop bulge within the ciliary membrane before internalization.[9][10]

After the cilium is retracted, the cyst germinates and generates a germ tube. The germ tube is then extended to form the rhizoidal system. Finally, the cyst develops into a sporangium, a reproductive structure, and mitosis begins. After five to eight times of synchronous mitosis, 32 – 256 zoospores form in the sporangium. Ciliogenesis probably occurs before cellularization. After cellularization, the zoospores escapes from the sporangium under suitable environmental condition.[3]

The timing of the cell cycle has been quantified using the S.punctatus expressing H2B-TdTomato controlled by H2B promoter under microscopy. The retraction of the cilium and the start of encysting happen within one hour. The germ tube appears in one to three hours. The first mitosis happens in eight to twelve hours. It finishes five to eight times of synchronous mitosis in thirty hours. The average cell cycle takes about 150 minutes. Each nuclear division is completed in 1 minute.[3]

Mitochondrial 5’ tRNA editing[]

This species is notable for having mitochondrial 5′ tRNA editing, a rare modification that is only known to also exist in the Amoebozoa species Acanthamoeba castellanii [1] and Chytridiomycota species Harpochytrium94, Harpochytrium105, Monoblepharella15, and Hyaloraphidium curvatum.[11][12] S. punctatus mitochondrial genome encodes eight tRNAs that recognize lysine, aspartic acid, tryptophan, methionine, tyrosine, glutamine, proline, and leucine codons. tRNALeu recognizes the UAG codon as leucine instead of the stop codon.[11]

tRNAs form secondary structures that are composed of helical stems. Predicted from mtDNA, mismatches are found in the first three nucleotides of the eight tRNA acceptor stems. Sequencing of the mature mitochondrial tRNAs showed the replacement of pyrimidines or purines by purines (A to G, U to G, U to A, and C to A) that restore the base pairing. The editing sites are always restricted to the first three positions.[11][13]

The mitochondrial 5’ tRNA editing of S.punctatus has been confirmed in vitro. Using mitochondrial extract, the 5’ mismatches of synthetic tRNA transcripts are removed and nucleotides are incorporated in a 3’ to 5’ direction by using the 3’ tRNA sequence as templates. The patterns of mitochondrial 5’ tRNA editing are similar to those found in A. castellanii.[14]

Phytohormone receptor homologs[]

Ethylene and cytokinin receptors in plants are histidine kinases.[15] Histidine kinases in fungi are hybrid histidine kinases due to the fusion of histidine kinase/histidine kinase-like ATPase catalytic domains (HK/HATPase domains) to the receiver domain. Ethylene and cytokinin receptor homologs are also found in several flagellated and unflagellated fungal genera, including Spizellomyces. In general, these two phytohormones are signaling molecules in plant biotic interactions. Ethylene and cytokinin receptors in early diversifying fungus may play important roles in colonizing land.[2]

Opsins[]

Belonging to Claas A family of G-protein coupled receptors, opsins are seven-transmembrane proteins with photoreception functions.[16] Opsins bind to retinylidene compounds to form rhodopsins, which function as light sensors. There are two types of rhodopsins. Type 1 rhodopsins are found in bacteria, while type 2 rhodopsins are present in metazoan. Template-based structure modeling suggests that S. punctatus opsin is structurally similar to animal type 2 rhodopsins. It forms a stable structure when binding to retinaldehyde chromophore. In contrast, Dikarya rhodopsins are type 1 forms. In Blastocladiella emersonii, a flagellated early-diverging fungus, type 1 rhodopsin is responsible for phototaxis. However, the biological function of S. punctatus opsin is unexplored.[17]

References[]

  1. ^ a b Russ C, Lang BF, Chen Z, Gujja S, Shea T, Zeng Q, et al. (August 2016). "Genome Sequence of Spizellomyces punctatus". Genome Announcements. 4 (4). doi:10.1128/genomeA.00849-16. PMC 4991717. PMID 27540072.
  2. ^ a b Hérivaux A, Dugé de Bernonville T, Roux C, Clastre M, Courdavault V, Gastebois A, et al. (January 2017). Taylor JW (ed.). "The Identification of Phytohormone Receptor Homologs in Early Diverging Fungi Suggests a Role for Plant Sensing in Land Colonization by Fungi". mBio. 8 (1). doi:10.1128/mBio.01739-16. PMC 5285503. PMID 28143977.
  3. ^ a b c d e Medina EM, Robinson KA, Bellingham-Johnstun K, Ianiri G, Laplante C, Fritz-Laylin LK, Buchler NE (May 2020). "Genetic transformation of Spizellomyces punctatus, a resource for studying chytrid biology and evolutionary cell biology". eLife. 9: e52741. doi:10.7554/eLife.52741. PMC 7213984. PMID 32392127.
  4. ^ Fisher MC, Garner TW (June 2020). "Chytrid fungi and global amphibian declines". Nature Reviews. Microbiology. 18 (6): 332–343. doi:10.1038/s41579-020-0335-x. PMID 32099078. S2CID 211266075.
  5. ^ Koch WJ (May 1957). "Two new chytrids in pure culture, Phlyctochytrium punctatum and Phlyctochytrium irregulare". Journal of the Elisha Mitchell Scientific Society. 73 (1): 108–122. JSTOR 24333923.
  6. ^ Ruiz-Trillo I, Burger G, Holland PW, King N, Lang BF, Roger AJ, Gray MW (March 2007). "The origins of multicellularity: a multi-taxon genome initiative". Trends in Genetics. 23 (3): 113–118. doi:10.1016/j.tig.2007.01.005. PMID 17275133.
  7. ^ Russ C, Lang BF, Chen Z, Gujja S, Shea T, Zeng Q, et al. (August 2016). "Genome Sequence of Spizellomyces punctatus". Genome Announcements. 4 (4). doi:10.1128/genomeA.00849-16. PMC 4991717. PMID 27540072.
  8. ^ Swafford AJ, Hussey SP, Fritz-Laylin LK (September 2020). "High-efficiency electroporation of chytrid fungi". Scientific Reports. 10 (1): 15145. Bibcode:2020NatSR..1015145S. doi:10.1038/s41598-020-71618-2. PMC 7493940. PMID 32934254.
  9. ^ Koch WJ (1968). "Studies of the Motile Cells of Chytrids. V. Flagellar Retraction in Posteriorly Uniflagellate Fungi". American Journal of Botany. 55 (7): 841–859. doi:10.1002/j.1537-2197.1968.tb07442.x. ISSN 1537-2197.
  10. ^ Venard CM, Vasudevan KK, Stearns T (October 2020). "Cilium axoneme internalization and degradation in chytrid fungi". Cytoskeleton. 77 (10): 365–378. doi:10.1002/cm.21637. PMC 7944584. PMID 33103844.
  11. ^ a b c Laforest MJ, Bullerwell CE, Forget L, Lang BF (August 2004). "Origin, evolution, and mechanism of 5' tRNA editing in chytridiomycete fungi". RNA. 10 (8): 1191–1199. doi:10.1261/rna.7330504. PMC 1370609. PMID 15247432.
  12. ^ Bullerwell CE, Lang BF (August 2005). "Fungal evolution: the case of the vanishing mitochondrion". Current Opinion in Microbiology. Host--microbe interactions: fungi / edited by Howard Bussey · Host--microbe interactions: parasites / edited by Artur Scherf · Host--microbe interactions: viruses / edited by Margaret CM Smith. 8 (4): 362–369. doi:10.1016/j.mib.2005.06.009. PMID 15993645.
  13. ^ Laforest MJ, Roewer I, Lang BF (February 1997). "Mitochondrial tRNAs in the lower fungus Spizellomyces punctatus: tRNA editing and UAG 'stop' codons recognized as leucine". Nucleic Acids Research. 25 (3): 626–632. doi:10.1093/nar/25.3.626. PMC 146481. PMID 9016605.
  14. ^ Bullerwell CE, Gray MW (January 2005). "In vitro characterization of a tRNA editing activity in the mitochondria of Spizellomyces punctatus, a Chytridiomycete fungus". The Journal of Biological Chemistry. 280 (4): 2463–2470. doi:10.1074/jbc.M411273200. PMID 15546859.
  15. ^ Bidon B, Kabbara S, Courdavault V, Glévarec G, Oudin A, Héricourt F, et al. (November 2020). "Cytokinin and Ethylene Cell Signaling Pathways from Prokaryotes to Eukaryotes". Cells. 9 (11): 2526. doi:10.3390/cells9112526. PMC 7700396. PMID 33238457.
  16. ^ Katritch V, Cherezov V, Stevens RC (2013-01-06). "Structure-function of the G protein-coupled receptor superfamily". Annual Review of Pharmacology and Toxicology. 53 (1): 531–556. doi:10.1146/annurev-pharmtox-032112-135923. PMC 3540149. PMID 23140243.
  17. ^ Ahrendt SR, Medina EM, Chang CA, Stajich JE (2017-04-27). "Exploring the binding properties and structural stability of an opsin in the chytrid Spizellomyces punctatus using comparative and molecular modeling". PeerJ. 5: e3206. doi:10.7717/peerj.3206. PMC 5410147. PMID 28462022.
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